The hepatitis C virus (HCV) is the major causative agent of parenterally-transmitted and sporadic non-A, non-B hepatitis (NANB-H). Some 1% of the human population of the planet is believed to be affected. Infection by the virus can result in chronic hepatitis and cirrhosis of the liver, and may lead to hepatocellular carcinoma. Currently no vaccine nor established therapy exists, although partial success has been achieved in a minority of cases by treatment with recombinant interferon-α, either alone or in combination with ribavirin. There is therefore a pressing need for new and broadly-effective therapeutics.
Several virally-encoded enzymes are putative targets for therapeutic intervention, including a metalloprotease (NS2-3), a serine protease (N-terminal part of NS3), a helicase (C-terminal part of NS3), and an RNA-dependent RNA polymerase (NS5B). The NS3 protease is located in the N-terminal domain of the NS3 protein, and is considered a prime drug target since it is responsible for an intramolecular cleavage at the NS3/4A site and for downstream intermolecular processing at the NS4A/4B, NS4B/5A and NS5A/5B junctions.
Previous research has identified classes of peptides, in particular hexapeptides, showing degrees of activity in inhibiting the NS3 protease. The aim of the present invention is to provide further compounds which exhibit similar, and if possible improved, activity.
According to the nomenclature of Schechter & Berger (1967, Biochem. Biophys. Res. Commun. 27, 157–162) cleavage sites in substrates for the NS3 protease are designated P6-P5-P4-P3-P2-P1 . . . P1′-P2′-P3′-P4′-, with each P representing an amino acid, and the scissile bond lying between P1 and P1′. Corresponding binding sites on the enzyme are indicated as S6-S5-S4-S3-S2-S1 . . . S1′-S2′-S3′-S4′.
The present applicant has previously disclosed so called product inhibitors which are based on the P-region of the natural cleavage sites and which have been optimised to low nanomolar potency ((1998) Biochemistry 37: 8899–48905 and (1998) Biochemistry 37: 8906–8914. These inhibitors extract much of their binding energy from the C-terminal carboxylate, the remaining interactions with NS3 being similar to the ones used by the natural substrates, including binding in the S1 pocket and the prominent electrostatic interaction of the P6-P5 acidic couple.
At variance with the P region, the P′ region of the substrate, while being important for catalysis, does not influence significantly ground-state binding to the enzyme as expressed by the Km value. In other words, binding energy released by the substrate interaction with the enzyme to form an initial non-covalent complex is essentially due to the interaction of the residues of the P region; the P′ region residues contribute to a lesser extent to the binding energy. Accordingly, peptides based on the P′ region of the natural substrates (spanning residues P1′–P10′) do not inhibit NS3 to any significant extent. This notwithstanding, inspection of the crystal structure of NS3 with or without 4A (and more recently of the NMR structure of NS3) shows the presence of binding pockets in the S′ region which might be exploited for the binding of active-site directed inhibitors. S′-binding ligands would therefore display a range of interactions with the enzyme different from the ones used by the substrate, and represent a novel class of NS3 inhibitors.
Landro et al in (1997) Biochemistry 36, 9340–9348 synthesized certain non-cleavable decapeptides based on the NS5A/5B cleavage site by substituting the P1′ serine by a bulky cyclic aromatic (tetrahydroisoquinoline-3carboxylic acid) or smaller cyclic alkyl compound (proline or pipecolinic acid). They then investigated the interaction of these decapeptides with the substrate binding site of NS3 either in the presence or absence of NS4A cofactor. By looking at the effect of truncation at either the P or P′ side of the molecule they concluded that most of the binding energy of the decapeptide is due to interactions with NS3–NS4A complex on the P side of the molecule. Truncation on the P′ side produced a relatively large effect in the presence of NS4A cofactor, but less when NS4A was absent. They concluded that the P4′ substrate Tyr residue present in their molecules was in close proximity, or in direct contact with NS4A and that this residue contributes significantly to binding in the presence of NS4A.
WO-A-00/31129 describes the development of inhibitors which are more powerful than those described by Landro et al because they have better binding on their P′ side. In other words, the inhibitors take advantage of binding to the S′ region in addition to binding to the S-region of NS3. By varying the P′ amino acid residues, it was shown that the binding energy which may be extracted from S′-region binding is substantial, since inhibitors with optimised and non-optimised P′-regions differ in potency>1000-fold. Since no activity was present in any of the peptides corresponding to the isolated P′-region, optimisation of an S′-binding fragment was pursued in the context of non-cleavable decapeptides spanning P6–P4′.
Many of the compounds described in WO-A-00/31129 are oligopeptides, some including as many as ten amino acid residues. Peptidic molecules of this length may be unsuitable for peroral administration because they are subject to degradation in the digestive tract. Thus, it is desirable to develop shorter peptides or molecules having less peptidic character. With this in mind, the present inventors sought to develop a “collected product” inhibitor by combining an optimised P2′–P4′ fragment with a carboxylate positioned in the same way as the carboxylate in the P-region product inhibitors. They further sought to use the optimised P2′–P4′ fragment (cyclohexylalanine-aspartic acid-leucine-NH2) in the design of small molecular weight inhibitors also having a carboxylate appropriately positioned for binding to the enzyme.